When reactive substances such as proteins, peptides and amino acids having an amino group(s) coexist with a reducing sugar such as aldose having an aldehyde group(s), they combine non-enzymatically and irreversibly through the amino and aldehyde groups, which is followed by the amadori rearrangement to form an amadori compound. The production rate of an amadori compound being a function of the concentration of reactants, contacting period, temperature, and the like, various useful information about a sample containing such a reactive substance(s) can be derived from the amount of amadori compounds. Examples of materials containing an amadori compound include food products such as soy sauce and body fluids such as blood.
In a living body, fructosylamines are formed through the glycation reaction between glucose and various amino acids. For example, fructosylamines produced when hemoglobin and albumin in blood are glycated are called glycohemoglobin and glycoalbumin, respectively. The reducing ability of glycated derivative of a protein in blood is called "fructosamine". The concentration of these glycated derivatives in blood reflects an average level of blood sugar over a given period of time, and the determination thereof can be a significant index for diagnosis and control of conditions of diabetes. Thercfore, the establishment of a method of measuring an amadori compound in blood must be clinically very useful. Further, the state of preservation and period after production of a food product can be estimated on the basis of the amount of amadori compounds in the food product, and, therefore, the method of measuring an amadori compound also contributes to the quality control of a food product. As mentioned above, an assay of amadori compounds should be useful in a wide range of fields involving medicine and food products.
Examples of assays of amadori compounds include a method which utilizes high performance liquid chromatography [Chromatogr. Sci. 10: 659 (1979)], a column filled with solid materials to which boric acid is attached [Clin. Chem. 28: 2088-2094 (1982)], electrophoresis [Clin. Chem. 26: 1598-1602 (1980)] or antigen-antibody reaction [JJCLA 18: 620 (1993), J. Clin. Lab. Inst. Reag. 16: 33-37 (1993)], a method for measuring the fructosamine [Clin. Chim. Acta 127: 87-95 (1982)], a calorimetric determination following the oxidization with thiobarbituric acid [Clin. Chim. Acta 112: 197-204 (1981)], or the like. These existing methods, however, require an expensive device(s) and are not necessarily accurate and rapid enough.
In the field of clinical assay and food analysis, a method utilizing enzymatic process has become more and more popular, which makes it possible to analyze an intended substance selectively with accuracy and rapidity owing to characteristics of enzymes (specificity in terms of substrate, reaction, structure, active site, etc.)
There have been provided assays for determining amadori compounds on the basis of the amount of oxygen consumed or hydrogen peroxide generated in the reaction between an amadori compound and an oxidoreductase (e.g. Japanese Patent Publication (KOKOKU) Nos. 5-33997 and 6-65300, and Japanese Patent Publication (KOKAI) Nos. 2-195900, 3-155780, 4-4874, 5-192193 and 6-46846). Further, assays of glycated protein for the diagnosis of diabetes have also been proposed (Japanese Patent Publication (KOKAI) Nos. 2-195899, 2-195900, 5-192193 (EP-A-0526150), 6-46846 (EP-A-0576838).
The reaction between an amadori compound and an oxidoreductase can be represented by the following general formula: EQU R.sup.1 --CO--CH.sub.2 --NH--R.sup.2 +O.sub.2 +H.sub.2 O.fwdarw.R.sup.1 --CO--CHO+R.sup.2 --NH.sub.2 +H.sub.2 O.sub.2
wherein R.sup.1 is an aldose residue and R.sup.2 is an amino acid, protein or peptide residue.
Examples of enzymes which catalyze the above reaction are as follows:
1. Fructosyl amino acid oxidase derived from Corynebacterium (Japanese Patent Publication (KOKOKU) Nos. 5-33997 and 6-65300) or Aspergillus (Japanese Patent Publication (KOKAI) No. 3-155780)]; PA0 2. Fructosylamine deglycase derived from Candida (Japanese Patent Publication (KOKAI) No. 6-46846); PA0 3. Fructosyl amino acid deglycase derived from Penicillium (Japanese Patent Publication (KOKAI) No. 4-4874); PA0 4. Ketoamine oxidase derived from Corynebacterium, Fusarium, Acremonium or Debaryomyces (Japanese Patent Publication (KOKAI) No. 5-192193)]; and PA0 5. Alkyllysinase which can be prepared according to the method described in J. Biol. Chem., Vol. 239, pp. 3790-3796 (1964).
Assays involving these existing enzymes, however, have drawbacks. For instance, indexes for the diagnosis of diabetes in blood are glycated albumin, glycated hemoglobin and fructosylamine. Glycated albumin is formed when glucose is bound to a lysine residue at its e-position in a protein [J. Biol. Chem., 261: 13542-13545 (1986)]. In the case of glycated hemoglobin, glucose is also bound to the N-terminal valine residue of .beta.-chain in addition to a lysine residue [J. Biol. Chem. 254: 3892-3898 (1979)]. Therefore, it is necessary to use an enzyme more specific for fructosyl lysine compared to fructosyl valine in the determination of glycated proteins as an index of diabetes. However, an enzyme derived from Corynebacterium does not act on fructosyl lysine. The enzyme from Aspergillus disclosed in Japanese Patent Publication (KOKAI) No. 3-155780 is less active on fructosyl lysine than fructosyl valine and is not known about the action on glycated proteins or hydrolyzed products thereof. The ketoamine oxidase described in Japanese Patent Publication (KOKAI) No. 5-192193 can act on fructosyl valine, but does not afford an accurate assay of glycated proteins comprising a lysine residue bound to a sugar. Because the fructosylamine deglycase is highly specific for di-fructosyl lysine, it is inadequate for determining specifically a substance in which a lysine residue is glycated at the .epsilon.-position and/or a valine residue is glycated. Furthermore, a method using an alkyllysinase would be unreliable or inaccurate because the said enzyme is non-specific and reacts with substances comprising a lysine residue associated with a non-sugar moiety. The enzyme from Penicillium (Japanese Patent Publication (KOKAI) No. 4-4874) reacts on both of fructosyl lysine and fructosyl alanine.
As described above, the existing enzymes cannot necessarily give an accurate assay of intended glycated proteins and therefore the development of an enzyme more specific for fructosyl lysine compared to fructosyl valine has been demanded.
In general, for the improvement of accuracy and usefulness of an assay involving an enzymatic process, it is essential to use an enzyme having a catalytic activity suitable for the purposes of a given assay. Thus, it is necessary to select an appropriate enzyme taking many factors such as the substance (i.e., substrate) to be determined, the condition of the sample, measuring conditions, and the like, into consideration in order to carry out the assay with accuracy and reproducibility. To select such an enzyme, it is required that many enzymes are provided and characterized in advance.